宁波市腹泻患者隐孢子虫分子鉴定及基因特征分析

doi:10.12140/j.issn.1000-7423.2023.02.012

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (2): 202-208.doi: 10.12140/j.issn.1000-7423.2023.02.012

宁波市腹泻患者隐孢子虫分子鉴定及基因特征分析

秦源¹(), 刘华¹, 姜岩岩¹, 曹建平¹^,², 沈玉娟¹^,²^,^*()

1 中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心），国家卫生健康委员会寄生虫病原与媒介生物学重点实验室，世界卫生组织热带病合作中心，国家级热带病国际联合研究中心，上海 200025
2 上海交通大学医学院-国家热带病研究中心全球健康学院，上海 200025

收稿日期:2023-01-09 修回日期:2023-02-28 出版日期:2023-04-20 发布日期:2023-04-20
通讯作者: 沈玉娟
作者简介:秦源（1996-），女，硕士研究生，从事肠道原虫分子流行病学研究。E-mail：qy_1226@163.com
基金资助:
国家自然科学基金(82072307);国家自然科学基金(81772224);国家科技重大专项(2018ZX10713001-004)

Molecular identification and genetic characterization of Cryptosporidium spp. in patients with diarrhea in Ningbo City

QIN Yuan¹(), LIU Hua¹, JIANG Yanyan¹, CAO Jianping¹^,², SHEN Yujuan¹^,²^,^*()

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); NHC Key Laboratory of Parasite and Vector Biology; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, China
2 School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2023-01-09 Revised:2023-02-28 Online:2023-04-20 Published:2023-04-20
Contact: SHEN Yujuan
Supported by:
Support by the National Natural Science Foundation of China(82072307);Support by the National Natural Science Foundation of China(81772224);National Science and Technology Major Program of China(2018ZX10713001-004)

摘要/Abstract

摘要： 目的通过了解宁波市某哨点医院临床腹泻患者隐孢子虫感染情况及基因特征分析，为临床探究腹泻病因提供新思路。方法 2019年11月—2021年7月，收集宁波市某哨点医院临床腹泻患者粪样650份，提取粪样中基因组DNA，巢式PCR扩增隐孢子虫核糖体小亚基RNA（SSU rRNA）基因，产物经1.5%琼脂糖凝胶电泳检测。扩增出条带的阳性粪样DNA进行60-kDa糖蛋白（gp60）基因的巢氏PCR扩增，产物经1.5%琼脂糖凝胶电泳检测。将与目的条带大小相近的PCR扩增产物进行双向测序，根据GenBank公共数据库资源行BLAST分析，使用Clustal X 2.1软件比对和校准，根据序列分析结果进行隐孢子虫虫种鉴定和亚型分型。运用Mega 11.0软件，以neighbour-joining（N-J）法绘制系统进化树。结果收集的粪样中男性腹泻患者369份（56.8%），女性腹泻患者281份（43.2%）；年龄分布为1~99岁。经SSU rRNA基因扩增，仅1份DNA样品在830 bp左右出现特异性条带，呈隐孢子虫阳性。患者为53岁的男性，农民，水样便。经核苷酸序列分析，鉴定为火鸡隐孢子虫感染，与上海市人源分离株、黑龙江省奶牛源分离株、云南省鸡源分离株等的序列同源性为100%，系统进化树均落入同一分支。经gp60基因扩增，在955 bp左右出现特异性条带，鉴定为Ⅲb亚型家族，与人源ⅢbA24G1R1亚型的同源性最高（99.8%）；经序列比对，为火鸡隐孢子虫新亚型ⅢbA26G1（GenBank登录号：OQ032548）。结论宁波地区临床腹泻患者中鉴定到人兽共患的火鸡隐孢子虫，提示应加强腹泻人群隐孢子虫的监测和流调溯源。

关键词: 临床腹泻患者, 隐孢子虫, 火鸡隐孢子虫, 基因亚型

Abstract: Objective To analyze the Cryptosporidium infection and genetic characterization of outpatients with diarrhea in a sentinel hospital in Ningbo, to provide new ideas for clinical investigation on etiology of diarrhea. Methods A total of 650 fecal samples were collected from clinical diarrhea patients in NingBo City from November 2019 to July 2021. The genomic DNA from these samples was extracted, and the small subunit ribosomal ribonucleic acid gene (SSU rRNA) of Cryptosporidium was amplified by nested PCR. The products were analyzed by 1.5% agarose gel electrophoresis. The DNA from above positive sample was used as the template to amplify the 60-kDa glycoprotein (gp60) gene by nested PCR, of which the product was analyzed by 1.5% agarose gel electrophoresis. PCR amplification products of similar size to the target bands were sequenced in both directions, and blasted to identify the species or genotype according to the GenBank public database resources using Clustalx 2.1 software. Use Mega 11.0 software to draw the phylogenetic tree by the neighbour-joining (N-J) method. Results Among the fecal samples collected, 369 (56.8%) were from male diarrhea cases, and 281 (43.2%) female cases; of them the ages ranged from 1 to 99 years. After amplifying of the SSU rRNA gene, only one DNA sample showed specific bands at around 830 bp in electrophoresis, indicating Cryptosporidium spp. positive. The patient was a 53-year-old male, farmer with watery stool. According to the analysis of DNA sequence, it was identified as C. meleagridis, which has 100% homology with those of a human-derived isolate from Shanghai, a dairy-derived isolate from Heilongjiang Province, a chicken-derived isolate from Yunnan Province. The phylogenetic tree showed that these isolates locate in the same branch. The product of gp60 gene amplification displayed a specific band at around 955 bp, which was identified as the Ⅲb subtype family, with the highest homology (99.8%) to the human derived ⅢbA24G1R1 subtype, sequence alignment indicate it is a new subtype ⅢbA26G1 of C. meleagridis (GenBank accession No. OQ032548). Conclusion Zoonotic C. meleagridis was identified from a clinical diarrhea case in Ningbo area, suggesting that surveillance on Cryptosporidium infection in people with diarrhea and epidemiological tracing for infection source should be strengthened.

Key words: Clinical diarrhea patient, Cryptosporidium spp., C. meleagridis, Subtype

中图分类号:

R382.3

秦源, 刘华, 姜岩岩, 曹建平, 沈玉娟. 宁波市腹泻患者隐孢子虫分子鉴定及基因特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(2): 202-208.

QIN Yuan, LIU Hua, JIANG Yanyan, CAO Jianping, SHEN Yujuan. Molecular identification and genetic characterization of Cryptosporidium spp. in patients with diarrhea in Ningbo City[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(2): 202-208.

图/表 6

表1

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图2

图3

图4

表2

我国人源、动物源、水源中火鸡隐孢子虫分布情况

来源 Source	地区 Area	年份 Year	检出率/%（n/N） Rate of positive/% (n/N)	基因亚型（n） Subtype (n)	参考文献 References
人 Human
HIV/AIDS患者 HIV/AIDS patiens	河南 Henan	2006	-	-	[14]
		2007—2010	0.7 (5/683)	ⅢbA27G1R1 (1), ⅢbA29G1R1 (1), ⅢbA26G1R1 (1), ⅢeA26G2R1 (1), N^*(1)	[15]
	台湾 Taiwan	1994—2004	0.1 (1/1 044)	-	[16]
腹泻儿童 Diarrhea children	湖北 Hubei	2016	2.0 (10/500)	ⅢbA21G1R1 (1), ⅢbA22G1R1 (8), ⅢbA26G1R1 (1)	[17]
腹泻儿童 Diarrhea children	上海 Shanghai	2007—2009	0.1 (4/3 245)	-	[18]
腹泻成人 Diarrhea adults	浙江 Zhejiang	2019—2021	0.2 (1/571)	ⅢbA26G1 (1)	本研究 This study
动物 Animal
鹌鹑 Quail	河南 Henan	2006—2007	0.1 (2/1 818)	-	[19]
宠物鸟 Pet bird	河南 Henan	2018—2019	0.8 (8/1 005)	-	[20]
狐狸 Fox	吉林 Jilin	2014—2016	0.5 (1/213)	-	[21]
	山东 Shandong	2019	21.7 (65/299)	ⅢeA21G2R1 (20), ⅢeA19G2R1 (6), ⅢeA17G2R1 (1)	[22]
鸡 Chicken	安徽 Anhui	-	19.5 (39/200)	-	[23]
		2018—2019	0.2 (2/829)	ⅢbA26G1R1b (2)	[24]
	广东 Guangdong	2020—2021	7.8 (78/1 001)	ⅢgA25G3R1a (2), ⅢbA23G1R1c (24), ⅢbA24G1R1 (20), ⅢbA21G1R1a (7), ⅢeA17G2R1 (8), ⅢeA26G2R1 (3)	[25]
	河南 Henan	2006—2007	0.2 (3/1 542)	-	[26]
		2011	10.5 (25/238)	-	[27]
	湖北 Hubei	2017	3.2 (15/471)	ⅢbA22G1R1c (6), ⅢbA23G1R1d (2), ⅢbA26G1R1b (1), ⅢeA17G2R1 (1), ⅢeA19G2R1 (1)	[28]
	新疆 Xinjiang	2020—2021		ⅢbA23G1R1 (1), ⅢbA25G1R1 (20), ⅢbA31G1R1 (4)	[29]
		2022	0.2 (35/175)	ⅢgA22G3R1 (1), ⅢbA25G1R1 (10), ⅢgA24G3R1 (9), ⅢgA25G2R1 (1)	[30]
	云南 Yunnan	2020	-	-	[31]
	浙江 Zhejiang	2010—2012	1.0 (4/385)	-	[32]
牛 Catttle	黑龙江 Heilongjiang	2009—2011	3.3 (5/151）	ⅢeA22G2R1 (5)	[33]
水貂 Mink	吉林、辽宁、黑龙江 Jilin, Liaoning, Heilongjiang	2014	1.9 (3/162)	-	[34]
水 Water
河/湖水 River/lake water	上海Shanghai	2013—2014	2.3 (4/178)	ⅢbA24G1 (1)	[35]
	天津 Tianjin	-	1.9 (1/52)	-	[36]
	重庆 Chongqing	2011	1.5 (1/66)	-	[37]
污水 Sewage	广东 Guangdong	2018—2019	1.8 (6/326)	-	[38]
	南京 Nanjing	2007—2009	2.3 (2/87)	-	[39]
	青岛 Qingdao	2008	6.4 (7/109)	-	[39]
	上海 Shanghai	2006—2007	1.1 (7/90)	-	[40]
		2006—2007	7.8 (7/90)	-	[39]
		2012—2014	5.0 (4/80)	ⅢbA22G1R1c (1)	[41]
	武汉 Wuhan	2008	6.0 (6/100)	-	[39]
自来水 Tap-water	上海 Shanghai	2009—2010	1.3 (1/80)	-	[42]

表2

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位点 Locus	引物名称 Primer name	引物序列（5'→3'） Primer sequences（5'→3'）	产物长度/bp Product length/bp
SSU rRNA	Cry18sF1	TTCTAGAGCTAATACATGCG	1 100
	Cry18sR1	CCCATTTCCTTCGAAACAGGA	1 100
	Cry18sF2	GGAAGGGTTGTATTTATTAGATAAAG	830
	Cry18sR2	AAGGAGTAAGGAACAACCTCCA	830
gp60	F1	GATGAGATTGTCGCTCGTTATC	1 161
	R1	AACCTGCGGAACCTGTG	1 161
	F2	GAGATTGTCGCTCGTTATCG	955
	R2	GATTGCAAAAACGGAAGG	955

宁波市腹泻患者隐孢子虫分子鉴定及基因特征分析

Molecular identification and genetic characterization of Cryptosporidium spp. in patients with diarrhea in Ningbo City

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